Detection units and methods for detecting a target analyte

ABSTRACT

The present application relates to detection units and methods for detecting one or more target analytes in a sample using a complex formed by a target and first and second probes, wherein the first probe is coupled to a detectable piece, the target is coupled to the first probe and the second probe, and the second probe is coupled to a solid support. Specific binding of the detectable piece to the target analyte can be distinguished from non-specific binding of the detectable piece by measuring the number of detectable pieces that leave their initial location after exposure to a disruptor that uncouples the detectable piece from the solid support.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 16/420,231 filed May 23, 2019, now allowed, which claims thebenefit of U.S. Provisional Application No. 62/676,439 filed May 25,2018, the contents of which are incorporated by reference herein intheir entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grants No.R44AI122527, R43AG056208 and R43AI124871 awarded by the NationalInstitutes of Health. The U.S. government has certain rights in theinvention.

FIELD OF THE INVENTION

The present invention relates generally to detection units and methodsfor detecting a target analyte such as natural, synthetic, modified orunmodified nucleic acids or proteins in a sample.

BACKGROUND OF THE INVENTION

Many detection systems for determining the presence or absence of aparticular target analyte in a sample are known. Examples of detectionsystems for detecting analytes include immunoassays, such as an enzymelinked immunosorbent assays (ELISAs), which are used in numerousdiagnostic, research and screening applications. Generally, thesedetection systems detect the target analyte when it binds to a specificbinding agent or probe resulting in a measurable signal.

When using known detection systems, such as immunoassays, the ability todetect a target analyte is often limited by the low concentration of thetarget analyte in the sample and by non-specific interactions, such asnon-specific binding of signal producing molecules and non-specificbinding of sample molecules. The ability to detect a target analyte in abiological sample is often limited by these two factors.

The signal generated by detection systems is normally proportional tothe number of target analytes that bind to the specific binding probe.Therefore, when the concentration of target is low, the signal is low.The total signal can be increased by increasing the signal associatedwith each bound target analyte. Often, detection systems use a solidsupport and reporter markers, such as fluorescent molecules, to generatethe signal. Several strategies that use reporter markers have beendesigned to increase the signal associated with each bound target, suchas in branched-DNA (Hendricks et al., Am J Clin Pathol. 1995,104(5):537) and hybrid capture (WO 2003078966 A2). While thesestrategies increase the total signal, they often also increase thebackground noise resulting from the non-specific interaction between thereporter marker and the solid support. These strategies do not offer aneffective method of discriminating reporter markers non-specificallybound to the solid support.

The use of micrometer scale particles as reporter markers, described inPCT/GB2010/001913, offers a method to remove particles non-specificallybound to the solid support by applying a controlled fluid drag force onthe particles.

Another strategy, disclosed in PCT/GB2010/001913 (WO 2011/045570 A2),uses a magnetic bead tethered to a solid support by an elongatedmolecule as a sensing apparatus to detect, for example a signal from anELISA assay. According to this disclosure, reporter molecules arecleaved off the probes that bind to the target molecule on thesubstrate. The signal is amplified by releasing manipulating agents thatact on an elongated molecule that tethers a bead to the solid support ina separate compartment. The bead is tethered to the solid supportindependently of the presence or absence of the target analyte.

Accordingly, there is a need for detection units as well as methodscapable of detecting low concentrations of target analytes whiledistinguishing non-specific binding from specific binding in the sample.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method of detecting atarget analyte in a sample, the method comprising:

-   -   a) providing at least one detectable piece coupled to a solid        support via a complex formed by the target analyte and a first        and a second probe, wherein:        -   i) the first probe is coupled to the detectable piece and            bound to said analyte if present,    -   and        -   ii) the second probe is coupled to the solid support and            bound to said analyte if present, so that only if the target            analyte is present in the sample, the detectable piece is            directly or indirectly coupled to the solid support at an            initial location via the complex, wherein the complex            comprises an elongated region that is at least 100            nanometers in length;    -   b) either applying a force to the detectable piece and measuring        the displacement of the detectable piece or measuring the amount        of Brownian motion of the detectable piece;    -   c) exposing the complex to a disruptor that is capable of        uncoupling the detectable piece from the solid support;    -   d) optionally applying a force to the detectable piece; and    -   e) detecting if the detectable piece has left its initial        location;

wherein the presence of the target in the sample is indicated bydetectable pieces that: i) suffer a displacement or Brownian motionwithin a pre-determined range and ii) leave their initial location.

In another aspect, the present invention provides a method of detectinga target analyte in a sample, the method comprising:

-   -   a) providing at least one detectable piece coupled to a solid        support via a complex formed by the target analyte and a first        and a second probe, wherein:        -   i) the first probe is coupled to the detectable piece and            bound to said analyte if present, and        -   ii) the second probe is coupled to the solid support and            bound to said analyte if present, so that only if the target            analyte is present in the sample, the detectable piece is            directly or indirectly coupled to the solid support at an            initial location via the complex,    -   b) optionally detecting the presence of the detectable piece;    -   c) exposing the complex to a disruptor that is capable of        uncoupling the detectable piece from the solid support, wherein        the disruptor comprises a strand-displacement molecule capable        of dissociating one or more nucleic acid duplexes formed between        the target and a probe, or the disruptor comprises a degradation        molecule capable of breaking one or more covalent bonds of the        target analyte;    -   d) optionally applying a force to the detectable piece; and    -   e) detecting if any of the detectable pieces have left their        initial location;        wherein a detectable piece that is indirectly bound to the        analyte is likely to leave their initial location, whereas a        detectable piece that is not indirectly bound to the analyte is        unlikely to leave their initial location.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A depicts a solid support (3) with two detectable pieces linked toit. Detectable piece (1) is linked to the solid support via anon-specific attachment (9) which is an attachment that takes placewithout the intervention of the target analyte. Detectable piece (2) islinked to the solid support via a complex formed from: i) a first probe(4) coupled to the detectable piece (2) and bound to the target analyte(6) and ii) a second probe (5) coupled to the solid support (3) and tothe target analyte (6). Detectable piece (2) is indirectly coupled tothe solid support at an initial location. Exposure to astrand-displacement molecule (7) that binds to the first probe (4)disrupts the bond between the first probe and the target analyte. As aresult, detectable piece (2) is not linked to the solid support and canmove away from its initial location by Brownian motion or by theapplication of a force. The fact that detectable piece (1) does notleave its initial location indicates that it is not specificallyattached to the solid support. The fact that detectable piece (2) leavesits initial location indicates that the target analyte is present in thesample.

FIG. 1B depicts a solid support (3) with two detectable pieces linked toit. Detectable piece (1) is linked to the solid support via anon-specific attachment (9). Detectable piece (2) is linked to the solidsupport via a complex formed from: i) a first probe (4) coupled to thedetectable piece (2) and bound to the target analyte (6) and ii) asecond probe (5) coupled to the solid support (3) and to the targetanalyte (6). Detectable piece (2) is indirectly coupled to the solidsupport at an initial location. Exposure to a degradation molecule (10)breaks one or more bonds in the target analyte dividing the originaltarget molecule (6) into a first fragment (13) and a second fragment(14). As a result, detectable piece (2) is not linked to the solidsupport and can move away from its initial location by Brownian motionor by the application of a force. The fact that detectable piece (1)does not leave its initial location indicates that its attachment to thesolid support is non-specific. The fact that detectable piece (2) leavesits initial location indicates that the target analyte is present in thesample.

FIG. 1C depicts a solid support (3) with two detectable pieces linked toit. Detectable piece (1) is linked to the solid support via anon-specific attachment (9). Detectable piece (2) is linked to the solidsupport via a complex formed from: i) a first probe (4) coupled to thedetectable piece (2) and bound to the target analyte (6) and ii) asecond probe (5) coupled to the solid support (3) and to the targetanalyte (6). Detectable piece (2) is indirectly coupled to the solidsupport at an initial location. Exposure to an interaction disruptionmolecule (11) disrupts at least one of the bonds between the targetanalyte (6) and the first and second probes. As a result, detectablepiece (2) is not linked to the solid support and can leave its initiallocation by Brownian motion or by the application of a force. The factthat detectable piece (1) does not leave its initial location indicatesthat its attachment to the solid support is non-specific. The fact thatdetectable piece (2) leaves its initial location indicates that thetarget analyte is present in the sample.

FIG. 1D depicts a solid support (3) with two detectable pieces linked toit. Detectable piece (1) is linked to the solid support via anon-specific attachment (9). Detectable piece (2) is linked to the solidsupport via a complex formed from: i) a first probe (4) coupled to thedetectable piece (2) and bound to the target analyte (6) and ii) asecond probe (5) coupled to the solid support (3) and to the targetanalyte (6). Detectable piece (2) is indirectly coupled to the solidsupport at an initial location. Exposure to an interaction disruptionbuffer (12) disrupts at least one of the bonds between the targetanalyte (6) and the first (4) and second (5) probes. As a result,detectable piece (2) is not linked to the solid support and can moveaway from its initial location by Brownian motion or by the applicationof a force. The fact that detectable piece (1) does not leave itsinitial location indicates that its attachment to the solid support isnon-specific. The fact that detectable piece (2) leaves its initiallocation indicates that the target analyte is present in the sample.

FIG. 2A depicts a solid support (3) with three detectable pieces linkedto it. Detectable piece (201) is linked to the solid support via anon-specific attachment (9) to the solid support. Detectable piece (202)is linked to the solid support via a non-specific attachment (212) tothe second probe which is coupled to the solid support via theinteraction of region (209) with a probe (210) attached to the solidsupport. The second probe (205) comprises an elongated region.Detectable piece (202) is linked to the solid support without theintervention of the target analyte. Detectable piece (203) is linked tothe solid support via a complex formed from: i) a first probe (4)coupled to the detectable piece (203) and bound to the target analyte(6) and ii) a second probe (205) which comprises an elongated region andis coupled to the solid support (3) and to the target analyte (6). Thesecond probe (205) is coupled to the solid support via the interactionof region (209) with a probe (210) attached to the solid support.Measuring the displacement of detectable pieces (201), (202) and (203)under an external force will show that detectable piece (201) does notsuffer displacement, indicating that detectable piece (201) isnon-specifically attached to the solid support. However, it may not bepossible to differentiate the displacement of detectable piece (202) anddetectable piece (203) and therefore, it may not be possible to detectthat detectable piece (202) is non-specifically attached. Exposure to astrand-displacement molecule (7) that binds to the first probe (4)disrupts the bond between the first probe and the target analyte (itwill be recognized that the strand-displacement molecule (7) may alsobind to other locations within the complex (not shown) with the effectof releasing detectable piece (203) from solid support (3)). As aresult, detectable piece (203) is not linked to the solid support andleaves its initial location by Brownian motion or by the application ofa force. The fact that detectable piece (202) remains tethered to thesolid support indicates that its attachment is non-specific. The factthat detectable piece (203) leaves its initial location indicates thatthe target analyte is present in the sample.

FIG. 2B depicts a solid support (3) with three detectable pieces linkedto it as in FIG. 2A. Exposure to a degradation molecule (10) breaks oneor more bonds in the target analyte dividing the original targetmolecule (6) into a first fragment (13) and a second fragment (14). As aresult, detectable piece (203) is not linked to the solid support andleaves its initial location by Brownian motion or by the application ofa force. The fact that detectable piece (202) remains tethered to thesolid support indicates that its attachment is non-specific. The factthat detectable piece (203) leaves its initial location indicates thatthe target analyte is present in the sample.

FIG. 3 depicts the experiment described in Example 1.

FIG. 4A and FIG. 4B show the results of the experiment described inExample 1.

FIG. 5A and FIG. 5B show the results of the experiment described inExample 2.

FIG. 6 depicts the effect of the application of force on particlesattached to a solid support. If a particle (602) is attached to thesolid support via a complex that comprises an elongated region (as shownat (606)), the particle moves a distance (608) that is a function of thelength of the tether. If the particle (601) is non-specifically bound tothe solid surface (as shown at (607)), the particle does not move ormoves a distance significantly less than the specifically-bound particle(602).

DETAILED DESCRIPTION OF THE INVENTION

It should be appreciated that the particular implementations shown anddescribed herein are examples and are not intended to otherwise limitthe scope of the application in any way.

The published patents, patent applications, websites, company names, andscientific literature referred to herein are hereby incorporated byreference in their entirety to the same extent as if each wasspecifically and individually indicated to be incorporated by reference.Any conflict between any reference cited herein and the specificteachings of this specification shall be resolved in favor of thelatter. Likewise, any conflict between an art-understood definition of aword or phrase and a definition of the word or phrase as specificallytaught in this specification shall be resolved in favor of the latter.

As used in this specification, the singular forms “a,” “an” and “the”specifically also encompass the plural forms of the terms to which theyrefer, unless the content clearly dictates otherwise. The terms “about”and “substantially” are used herein to mean approximately, in the regionof, roughly, or around. When the terms “about” and “substantially” areused in conjunction with a numerical range, it modifies that range byextending the boundaries above and below the numerical values set forth.In general, the terms “about” and “substantially” are used herein tomodify a numerical value above and below the stated value by a varianceof less than about 20%.

Technical and scientific terms used herein have the meaning commonlyunderstood by one of skill in the art to which the present applicationpertains, unless otherwise defined. Reference is made herein to variousmethodologies and materials known to those of skill in the art. Standardreference works setting forth the general principles of recombinant DNAtechnology include Sambrook et al., “Molecular Cloning: A LaboratoryManual,” 2nd Ed., Cold Spring Harbor Laboratory Press, New York (1989);Kaufman et al., Eds., “Handbook of Molecular and Cellular Methods inBiology in Medicine,” CRC Press, Boca Raton (1995); and McPherson, Ed.,“Directed Mutagenesis: A Practical Approach,” IRL Press, Oxford (1991),the disclosures of each of which are incorporated by reference herein intheir entireties.

The terms “target analyte” or “analyte,” are used herein to denote themolecule to be detected in the test sample. According to the invention,there can be any number of different target analytes in the test sample(from one to one thousand, or even more). The target analyte can be anymolecule for which there exists a naturally or artificially preparedspecific binding member. Examples of target analytes include, but arenot limited to, a nucleic acid, oligonucleotide, DNA, RNA, protein,peptide, polypeptide, amino acid, antibody, carbohydrate, lipid,hormone, steroid, toxin, vitamin, any drug administered for therapeuticand illicit purposes, a bacterium, a virus, cell, as well as anyantigenic substances, haptens, antibodies, metabolites, water pollutants(such as nitrates, phosphates, heavy metals, etc.) and molecules havingan odor, such as compounds containing sulfur and/or nitrogen, forexample hydrogen sulfide, ammonia, amines, etc., and combinationsthereof.

In a preferred embodiment, the target analyte is a nucleic acid. Thenucleic acid can be from any source in purified or unpurified formincluding DNA (dsDNA and ssDNA) and RNA, including tRNA, mRNA, rRNA,siRNA, mitochondrial DNA and RNA, chloroplast DNA and RNA, DNA-RNAhybrids, or mixtures thereof, genes, chromosomes, plasmids, the genomesof biological materials, including microorganisms such as bacteria,yeast, viruses, viroids, molds, fungi, plants, animals, humans, andfragments thereof. The nucleic acid can be single stranded DNA obtainedby exposing double stranded DNA to an exonuclease enzyme, such asexonuclease III. The target analyte can be obtained from variousbiological materials by procedures well known in the art.

In another preferred embodiment, the target analyte is a short nucleicacid containing less than about 200 base pairs or less than about 200nucleotides. In general, such molecules are difficult to detect usingPCR-based techniques because suitable primers often cannot be found insuch a short sequence. A particular case of small DNA molecules aremolecules of less than about 40 nucleotides. These molecules are smallerthan the combined size of standard PCR primers (each primer about 20nucleotides). Short nucleic acid molecules are common in nature;exemplary cases are small interfering RNA (siRNA), micro-RNA (miRNA) andits precursors, pri-miRNA and pre-miRNA, and fragmented DNA moleculesproduced after cell death and present in blood, urine and other bodyfluids.

In another preferred embodiment, the target analyte is ribosomal RNA.Ribosomal ribonucleic acid (rRNA) is the RNA component of the ribosome,and is essential for protein synthesis in all living organisms. Itconstitutes the predominant material within the ribosome, which isapproximately 60% rRNA and 40% protein by weight, or ⅗ of ribosome mass.Ribosomes contain two major rRNAs and 50 or more proteins. The ribosomalRNAs form two subunits, the large subunit (LSU) and small subunit (SSU).The LSU rRNA acts as a ribozyme, catalyzing peptide bond formation. rRNAsequences are widely used for working out evolutionary relationshipsamong organisms, since they are of ancient origin and are found in allknown forms of life.

The term “probe” is understood herein to mean a molecule or a molecularcomplex formed by two or more molecules that is capable of binding tothe target analyte and also capable of being coupled to, depending onthe context, either to a solid support or to a detectable piece. Probeshave a region capable of binding to the target analyte.

The term “first probe” is understood herein to mean the probe that iscapable of coupling to a detectable piece. The term “second probe” isunderstood herein to mean the probe that is capable of coupling to thesolid support. For example, if the target analyte is a nucleic acid,oligonucleotide, DNA, or RNA, the region capable of binding the targetanalyte in both the first and second probe may comprise a nucleic acid,oligonucleotide, DNA, or RNA molecule having a sequence complementary tothe target analyte and capable of hybridizing thereto. As anotherexample, if the target analyte is a protein, peptide, polypeptide, oramino acid, the region capable of binding the target analyte in both thefirst and second probe may comprise an antibody, an antigen-bindingfragment or an aptamer that specifically binds to the target analyte.Probes may comprise regions of different nature. For example, a probemay comprise three different regions: a single stranded DNA region, adouble stranded DNA region and a protein region.

The terms “coupling”, “to couple”, “coupled”, “binding” “to bind”,“bound”, “link”, “linked”, “association”, “to attach” and “attachment”refer to any form of immobilization of a probe onto a surface, includingcovalent, non-covalent, direct, and mediated by one or more molecules.The same terms also refer to the covalent or non-covalent bondingbetween a probe and a target analyte. Non-covalent bonding can be formedby ionic interactions, via hydrogen bonding, etc.

The term “antibody” includes an immunoglobulin or an antigen-bindingfragment thereof.

The term “antigen-binding fragment” includes a part of an antibodymolecule that comprises amino acids responsible for the specific bindingbetween antibody and antigen. For certain antigens, the antigen-bindingdomain or antigen-binding fragment may only bind to a part of theantigen. The part of the antigen that is specifically recognized andbound by the antibody is referred to as the “epitope” or “antigenicdeterminant.” Antigen-binding domains and antigen-binding fragmentsinclude Fab (Fragment antigen-binding); a F(ab′)₂ fragment, a bivalentfragment having two Fab fragments linked by a disulfide bridge at thehinge region; Fv fragment; a single chain Fv fragment (scFv) see e.g.,Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883); a Fd fragment having the two V_(H)and C_(H)1 domains; dAb (Ward et al., (1989) Nature 341:544-546), andother antibody fragments that retain antigen-binding function. The Fabfragment has V_(H)—C_(H)1 and V_(L)—C_(L) domains covalently linked by adisulfide bond between the constant regions. The F_(v) fragment issmaller and has V_(H) and V_(L) domains non-covalently linked. Toovercome the tendency of non-covalently linked domains to dissociate, ascF_(v) can be constructed. The scF_(v) contains a flexible polypeptidethat links (1) the C-terminus of V_(H) to the N-terminus of V_(L), or(2) the C-terminus of V_(L) to the N-terminus of V_(H). A 15-mer(Gly4Ser)₃ peptide may be used as a linker, but other linkers are knownin the art. These antibody fragments are obtained using conventionaltechniques known to those with skill in the art, and the fragments areevaluated for function in the same manner as are intact antibodies.

The term “aptamer” includes oligonucleotide or peptide molecules thatbind to a specific target molecule. Aptamers are usually created byselecting them from a large random sequence pool, but natural aptamersalso exist in riboswitches. Aptamers can be used for both basic researchand clinical purposes as macromolecular drugs. Aptamers can be combinedwith ribozymes to self-cleave in the presence of their target molecule.These compound molecules have additional research, industrial andclinical applications. Nucleic acid aptamers are nucleic acid speciesthat have been engineered through repeated rounds of in vitro selectionor equivalently, SELEX (systematic evolution of ligands by exponentialenrichment) to bind to various molecular targets such as smallmolecules, proteins, nucleic acids, and even cells, tissues andorganisms (Molecules 2017, 22(2), 215). In the molecular level, aptamersbind to its target site through non-covalent interactions. Aptamers bindto these specific targets because of electrostatic interactions,hydrophobic interactions, and their complementary shapes. Aptamers areuseful in biotechnological and therapeutic applications as they offermolecular recognition properties that rival that of the commonly usedbiomolecule, antibodies. In addition to their discriminate recognition,aptamers offer advantages over antibodies as they can be engineeredcompletely in a test tube, are readily produced by chemical synthesis,possess desirable storage properties, and elicit little or noimmunogenicity in therapeutic applications. Peptide aptamers (Nature.380 (6574): 548) are artificial proteins selected or engineered to bindspecific target molecules. These proteins consist of one or more peptideloops of variable sequence displayed by a protein scaffold. They aretypically isolated from combinatorial libraries and often subsequentlyimproved by directed mutation or rounds of variable region mutagenesisand selection. In vivo, peptide aptamers can bind cellular proteintargets and exert biological effects, including interference with thenormal protein interactions of their targeted molecules with otherproteins.

The term “elongated region” refers to a section that may be part of thecomplex formed by the target analyte and the first and second probesthat is sufficiently long such that when the complex tethers adetectable piece to a solid support the displacement of the detectablepiece can be detected and differentiated from the displacement ofparticles that are non-specifically attached to the solid support. Inpreferred embodiments, the elongated region is a biomolecule, such as apolysaccharide, polypeptide or nucleic acid, longer than about 0.1micrometers, preferably between about 0.3 and about 100 micrometerslong. In even more preferred embodiments, the elongated region betweenabout 1 and about 50 micrometer long.

The terms “test sample” or “sample” are used interchangeably herein andinclude, but are not limited to, biological samples that can be testedby the methods of the present invention described herein and includehuman and animal body fluids such as whole blood, serum, plasma,cerebrospinal fluid, urine, lymph fluids, and various externalsecretions of the respiratory, intestinal and genitourinary tracts,tears, saliva, milk, white blood cells, myelomas and the like,biological fluids such as cell culture and blood culture, fixed tissuespecimens and fixed cell specimens, PCR amplification products or apurified product of one of the above samples. Additional relevantsamples of the present invention are lysates of body fluids and lysatesof other fluids that contain cells. In a lysate, all or some of thecells of the original fluid have been lysed. Many methods are known inthe art for lysing cells, including, sample heating, surfactants,enzymes and bead beating. A “sample” may include gaseous mediums, suchas ambient air, chemical or industrial intermediates, chemical orindustrial products, chemical or industrial byproducts, chemical orindustrial waste, exhaled vapor, internal combustion engine exhaust, orheadspace vapor such as vapor surrounding foods, beverages, cosmetics,vapor surrounding plant or animal tissue and vapor surrounding amicrobial sample. Another example of “sample” relevant to this inventionis a liquid solution produced by dissolving material collected byfiltering a gaseous sample or a liquid solution produced by exposing theliquid to a gaseous sample. Additional sample mediums includesupercritical fluids such as supercritical CO₂ extricate. Otherexemplary mediums include liquids such as water or aqueous solutions,oil or petroleum products, oil-water emulsions, liquid chemical orindustrial intermediates, liquid chemical or industrial products, liquidchemical or industrial byproducts, and liquid chemical or industrialwaste. Additional exemplary sample mediums include semisolid mediumssuch as animal or plant tissues, microbial samples, or samplescontaining gelatin, agar or polyacrylamide.

As used herein, a “detectable signal” which can be generated accordingto the invention includes, but is not limited to, an electrical,mechanical, optical, magnetic, acoustic or thermal signal. In preferredembodiments, the detectable signal is optical.

The term “solid support” is used herein to denote any solid materialsuitable for coupling to a probe and which is amenable to the detectionmethods disclosed herein. The number of possible suitable materials islarge and would be readily known by one of ordinary skill in the art.

The term “detectable region” is used to indicate a region of the solidsupport. The “detectable region” may in some cases be the entire solidsupport.

The term “detectable piece” is used to indicate a structure suitable forcoupling to a probe and which is amenable to the detection methodsdisclosed herein. The detectable piece can be directly or indirectlydetected. Examples of “detectable piece” of importance for the presentinvention include a particle, fluorescent particle, fluorescentmolecule, and enzymes that catalyze the formation of a detectablemolecule, such as horseradish peroxidase and alkaline phosphatase.

The term “disruptor” is used to indicate a molecule, a buffer,combinations thereof, or other chemical agents that alone or incombination with other conditions can uncouple a detectable piece fromthe solid support that is coupled to the solid support via a targetcomplex. Preferably, the disruptor will uncouple to a lesser extent adetectable piece that is coupled to the solid support withoutparticipation of the target analyte.

The association of the target and the first probe and the second probeforms a “complex” or “target complex”. The detectable piece can beindirectly coupled to the solid support via a target complex. Thedisruptor disrupts a target complex when the complex is broken up in twoor more parts such that it is not able to couple a detectable piece tothe solid support and/or when the coupling of the first probe to thedetectable piece is broken and/or when the coupling of the second probeto the solid support is broken. In preferred embodiments, the disruptoraction requires the presence of the target.

Examples of disruptor buffers are buffers where non-covalent attractionsbetween molecules are reduced and buffers where electrostatic repulsionsare increased. Important examples of disruptor buffers are buffers withlow salt, in which electrostatic repulsion increases, buffers with highsalt, in which ionic attraction is reduced, high pH buffers, and organicsolvents.

The term “degradation molecule” is used herein to indicate a disruptorthat breaks one or more covalent bonds.

The term “degradation” is used to indicate a process where one or morecovalent bonds are broken.

In preferred embodiments, the disruptor is a degradation molecule.Disruptors of this type are abundant and known to someone skilled in theart. Important examples are nucleases and proteases. The phosphodiesterbonds of nucleic acids can be broken using a nuclease enzyme. Nucleasescan generate single and double stranded breaks in their targetmolecules. In living organisms, they are essential machinery for manyaspects of DNA repair. Nucleases are also extensively used in molecularcloning. There are two primary classifications based on the locus ofactivity. Exonucleases digest nucleic acids from the ends. Endonucleasesact on regions in the middle of target molecules. They are furthersubcategorized as deoxyribonucleases and ribonucleases. The former actson DNA, the latter on RNA. Important examples for this application ofenzymes that act on RNA include: RNase H, RNase A, RNase III, RNase L,RNase P, RNase PhyM, RNase Ti, RNase T2, RNase U2, or RNase V. Importantexamples for this application of enzymes that act on DNA includerestriction endonucleases and nicking endonucleases. The peptide bondsof proteins and polypeptides can be broken using a protease. Someproteases detach the terminal amino acids from the protein chain(exopeptidases, such as aminopeptidases, carboxypeptidase A); othersattack internal peptide bonds of a protein (endopeptidases, such astrypsin, chymotrypsin, pepsin, papain, elastase). Proteolysis can behighly promiscuous such that a wide range of protein substrates arehydrolysed. This is the case for digestive enzymes such as trypsin.Promiscuous proteases typically bind to a single amino acid on thesubstrate and so only have specificity for that residue. For example,trypsin is specific for the sequences . . . K\ . . . or . . . R\ . . .(‘\’=cleavage site). Proteinase K is a broad-spectrum serine protease.The predominant site of cleavage is the peptide bond adjacent to thecarboxyl group of aliphatic and aromatic amino acids with blocked alphaamino groups. It is commonly used for its broad specificity. Conversely,some proteases are highly specific and only cleave substrates with acertain sequence. Blood clotting (such as thrombin) and viralpolyprotein processing (such as TEV protease) requires this level ofspecificity in order to achieve precise cleavage events. This isachieved by proteases having a long binding cleft or tunnel with severalpockets along it which bind the specified residues. For example, TEVprotease is specific for the sequence . . . ENLYFQ\S . . . (‘\’=cleavagesite).

In even more preferred embodiments, the disruptor is a degradationmolecule that can break one or more covalent bonds of the target analyteand does not break bonds of the probes. A detectable piece that isattached to the solid support without the target analyte(non-specifically) will not be uncoupled from the solid support by thistype of disruptor, while a detectable piece that is coupled to the solidsupport via the target complex will be readily uncoupled by this type ofdisruptor. Examples of this embodiment are the following: the targetanalyte is a protein, the probes are nucleic acid aptamers and thedegradation molecule is a proteinase enzyme. In another example, thetarget analyte is an RNA molecule, the probes are DNA molecules and thedegradation molecule is a ribonuclease enzyme.

FIG. 1B and FIG. 2B show examples of embodiments of the presentinvention where the disruptor breaks one or more bonds within the targetanalyte. FIG. 1B depicts a solid support (3) with two detectable pieceslinked to it. Detectable piece (1) is linked to the solid support via anon-specific attachment (9). Detectable piece (2) is linked to the solidsupport via a complex formed from: i) a first probe (4) coupled to thedetectable piece (2) and bound to the target analyte (6) and ii) asecond probe (5) coupled to the solid support (3) and to the targetanalyte (6). Detectable piece (2) is indirectly coupled to the solidsupport at an initial location. Exposure to a degradation molecule (10)breaks one or more bonds in the target analyte dividing the originaltarget molecule (6) into a first fragment (13) and a second fragment(14). As a result, detectable piece (2) is not linked to the solidsupport and can leave its initial location by Brownian motion or by theapplication of a force. The fact that detectable piece (1) does notleave its initial location indicates that its attachment to the solidsupport is nonspecific. The fact that detectable piece (2) leaves itsinitial location indicates that the target analyte is present in thesample.

FIG. 2B depicts a solid support (3) with three detectable pieces linkedto it. Detectable piece (201) is linked to the solid support via anon-specific attachment (9) to the solid support. Detectable piece (202)is linked to the solid support via a non-specific attachment (212) tothe second probe which is coupled to the solid support via theinteraction of region (209) with a probe (210) attached to the solidsupport. The second probe (205) comprises an elongated region.Detectable piece (202) is linked to the solid support without theintervention of the target analyte. Detectable piece (203) is linked tothe solid support via a complex formed from: i) a first probe (4)coupled to the detectable piece (203) and bound to the target analyte(6) and ii) a second probe (205) which comprises an elongated region andis coupled to the solid support (3) and to the target analyte (6). Thesecond probe (205) is coupled to the solid support via the interactionof region (209) with a probe (210) attached to the solid support.Measuring the displacement of detectable piece (201), (202) and (203)under an external force will show that detectable piece (201) does notsuffer displacement, indicating that detectable piece (201) isnon-specifically attached to the solid support. However, it may not bepossible to differentiate the displacement of detectable piece (202) anddetectable piece (203) and therefore, it may not be possible to detectthat detectable piece (202) is non-specifically attached. Exposure to adegradation molecule (10) breaks one or more bonds in the target analytedividing the original target molecule (6) into a first fragment (13) anda second fragment (14). As a result, detectable piece (203) is notlinked to the solid support and can move away from its initial locationby Brownian motion or by the application of a force. The fact thatdetectable piece (202) remains tethered indicates that its attachment isnon-specific. The fact that detectable piece (203) leaves its initiallocation indicates that the target analyte is present in the sample.

In other preferred embodiments, the disruptor is a molecule that breaksnon-covalent interactions. Examples of these disruptors arestrand-displacement molecules.

A “strand-displacement” disruptor molecule is a nucleic acid that aloneor in combination with other conditions denature a nucleic acid duplexinvolved in coupling the detectable piece to the solid support byhybridizing to one of the strands in the duplex. This type of reactionbetween nucleic acids is known as “strand displacement” (Zhang andWinfree, J. Am. Chem. Soc. 2009, 131, 17303). In a strand displacementreaction, a duplex formed by nucleic acid strands S1 and S2 is disruptedby a strand-displacement nucleic acid S3 that is complementary to S1. Inmost cases, S1 has a short single-stranded region, known as a toehold,that is not part of the nucleic acid duplex being disrupted (the toeholddoes not hybridize to S2). In most cases, during a strand displacementreaction S3 hybridizes first to the toehold in S1 and then proceeds tohybridize to the rest of S1. The presence of the toehold significantlyaccelerates the strand displacement reaction. Examples of howstrand-displacement nucleic acids can be used in this invention aredescribed below. The bond between the target analyte and the firstand/or the second probe may be broken using a strand displacementreaction. The strand-displacement molecule used in this reaction mayhybridize to the target, to the first probe or to the second probe anddisrupts the duplex formed by the target with one or both probes. FIG.1A, FIG. 2A and FIG. 3 show examples where the strand-displacementmolecule hybridizes to the first probe breaking the bond between thefirst probe and the target analyte. The sequence specificity of theassay can be increased using strand-displacement disruptors thathybridize to the target and use a toehold in the target molecule. Onlytarget-probe duplexes formed by targets that have the toehold sequencewill be disrupted. Another example of strand-displacement disruptors aredisruptors that disrupt the coupling between the first probe and thedetectable piece. For example, the first probe and a molecule M which isimmobilized to the detectable piece form a duplex and this nucleic acidduplex can be disrupted by strand displacement. In this case, thestrand-displacement molecule can hybridize to the first probe or tomolecule M. In any strand displacement reaction, the strand-displacementmolecule does not need to hybridize to the full region participating inthe duplex to be disruptive, instead, it may only need to displaceenough of one of the strands, so that the remainder of the duplex isunstable and denatures. In preferred embodiments, thestrand-displacement disruptor hybridizes to the target analyte anddisrupts the interaction with the first and/or the second probe, so thatit specifically uncouples detectable pieces that are coupled via thetarget analyte. In other preferred embodiments, the strand-displacementdisruptor hybridizes to the first or to the second probe and disruptsthe interaction of the first or second probe with the target analyte.

Other examples of disruptors that break non-covalent interactions aremolecules that disrupt non-covalent interactions of molecules in waterincluding electrostatic interactions, such as hydrogen bonding,π-effects, van der Waals forces and hydrophobic effects. This type ofdisruptor molecule may directly break the interaction between twomolecules or denature one or both of the molecules which leads to theirdissociation. An important type of disruptor in this category arechaotropic agents. A chaotropic agent is a molecule in water solutionthat can disrupt the hydrogen bonding network between water molecules(i.e. exerts chaotropic activity). This has an effect on the stabilityof the native state of other molecules in the solution, mainlymacromolecules (e.g., proteins and nucleic acids) by weakening thehydrophobic effect. For example, a chaotropic agent reduces the amountof order in the structure of a protein formed by water molecules, bothin the bulk and the hydration shells around hydrophobic amino acids andmay cause its denaturation. Common chaotropic agents of importance forthis invention include n-butanol, ethanol, guanidinium chloride, lithiumperchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol,sodium dodecyl sulfate, thiourea, formamide and urea.

In an embodiment of the present invention, two or more target analytesare detected. According to this embodiment, a different combination offirst and second probe is used for each target analyte, wherein eachtarget forms a different complex with the probes that bind to it. One ormore disruptors are used, wherein each of these disruptors disrupts thetarget complex of one or more target analytes. If two or more disruptorsare used, they can be put in contact with the target complexessimultaneously (all of them at the same time), or in a sequential manner(one or more at a time). The detectable pieces on the solid support aremonitored as each of the disruptors is used. The detectable pieces thatleave their location during exposure to a disruptor are indicative ofthe presence of the probe-target complex that the disruptor is capableof disrupting.

In an example of this embodiment, two or more strand-displacementdisruptors are used to detect multiple target analytes in the presentinvention. The target complexes are exposed to groups of one or morestrand-displacement disruptors in a sequential manner and the number ofdetectable pieces that leave their initial location after exposure toeach group of disruptors is determined.

The term “surface” or “surfaces” is used to indicate the external layerof the solid support and the particles. In exemplary embodiments, thesolid support or the particles may be composed of modified orfunctionalized glasses, inorganic glasses, plastics, including acrylics,polystyrene and copolymers of styrene, polypropylene, polyethylene,polybutylene, polyurethanes, Teflon, polysaccharides, nylon ornitrocellulose, resins, and other polymers, carbon, metals, ceramics,silica or silica-based materials including silicon and modified siliconand silicon wafers. In aspects, the surface can be a composite material.

Surfaces can be functionalized with molecules by physical or chemicaladsorption. In preferred embodiments, the surfaces are functionalizedwith probes or with molecules capable of coupling to probes. Suchmethods of functionalization are known in the art. For instance, a goldsurface can be functionalized with nucleic acids that have been modifiedwith alkanethiols at their 3′-termini or 5′-termini. See, for example,Whitesides, Proceedings of the Robert A. Welch Foundation 39thConference On Chemical Research Nanophase Chemistry, Houston, Tex.,pages 109-121 (1995). See also Mucic et al., Chem. Commun. 555-557(1996) (describes a method of attaching 3′ thiol DNA to flat goldsurfaces; this method can be used to attach oligonucleotides tonanoparticles). The alkanethiol method can also be used to attacholigonucleotides to other metal and semiconductors. Other functionalgroups for attaching oligonucleotides to solid surfaces includephosphorothioate groups (see, e.g., U.S. Pat. No. 5,472,881 for thebinding of oligonucleotide-phosphorothioates to gold surfaces),substituted alkylsiloxanes (see, e.g. Burwell, Chemical Technology, 4,370-377 (1974) and Matteucci and Caruthers, J. Am. Chem. Soc., 103,3185-3191 (1981) for binding of oligonucleotides to silica and glasssurfaces, and Grabar et al., Anal. Chem., 67, 735-743 for binding ofaminoalkylsiloxanes and for similar binding of mercaptoaklylsiloxanes)-.Oligonucleotides terminated with a 5′ thionucleoside or a 3′thionucleoside may also be used for attaching oligonucleotides to solidsurfaces. Another example of surface functionalization that is importantfor the present invention is the immobilization of antibodies and otherbinding members to the surface either by physical adsorption or bydirect or indirect chemical linkage. For instance, surfaces can befunctionalized by chemically linking streptavidin molecules to them,which are capable of coupling to probes comprising one or more biotinmolecules. The following reference describes the attachment of biotinlabeled oligonucleotides to a streptavidin functionalized surface. Shaiuet al., Nucleic Acids Research, 21, 99 (1993). Digoxigenin andanti-Digoxigenin antibodies can also be used to attach probes tosurfaces.

The surfaces can be functionalized by a monolayer of one or moremolecules. Methods of producing self-assembled monolayers are well knownin the art. In particular, there are several known methods to assemblemonolayers of thiolates on metal surfaces. See e.g., Love, J. C. et al.,Chem. Rev., 105, 1103 (2005).

The surface functionalization methods described above can be used tocouple molecules that prevent or reduce non-specific interactions withthe surface. For instance, after immobilization on to the surface of ananalyte binding molecule, such as a ssDNA or an antibody, physicaladsorption on the surface of a protein that blocks non-specificinteractions is often conducted. Common proteins used as blockers are:bovine serum albumin (BSA), fish serum and milk proteins, such ascasein.

The following references describe other methods that may be employed toattach oligonucleotides to surfaces: Nuzzo et al., J. Am. Chem. Soc.,109, 2358 (1987) (disulfides on gold); Allara and Nuzzo, Langmuir, 1, 45(1985) (carboxylic acids on aluminum); Allara and Tompkins, J. ColloidInterface Sci., 49, 410-421 (1974) (carboxylic acids on copper); Iler,The Chemistry Of Silica, Chapter 6, (Wiley 1979) (carboxylic acids onsilica); Timmons and Zisman, J. Phys. Chem., 69, 984990 (1965)(carboxylic acids on platinum); Soriaga and Hubbard, J. Am. Chem. Soc.,104, 3937 (1982) (aromatic ring compounds on platinum); Hubbard, Acc.Chem. Res., 13, 177 (1980) (sulfolanes, sulfoxides and otherfunctionalized solvents on platinum); Hickman et al., J. Am. Chem. Soc.,111, 7271 (1989) (isonitriles on platinum); Maoz and Sagiv, Langmuir, 3,1045 (1987) (silanes on silica); Maoz and Sagiv, Langmuir, 3, 1034(1987) (silanes on silica); Wasserman et al., Langmuir, 5, 1074 (1989)(silanes on silica); Eltekova and Eltekov, Langrnuir, 3, 951 (1987)(aromatic carboxylic acids, aldehydes, alcohols and methoxy groups ontitanium dioxide and silica); Lec et al., J. Phys. Chem., 92, 2597(1988) (rigid phosphates on metals).

Glass and other materials can be functionalized with a silane aldehyde,Triethoxysilylundecanal and the aldehyde-modified glass surface can thencovalently bind amino-containing biomolecules (J. Mater. Chem., 2011,21, 4384-4392; NATURE PROTOCOLS, 2006, 1, 1711-1724). Another importanttype of functionalization involves azides. The copper catalyzed Huisgen1,3 cycloaddition of azides and alkynes to form 1,2,3 triazoles (“click”chemistry) has become a very popular reaction in bioconjugation over thepast decades (Pharm Res. 2008 October; 25(10): 22162230; Drug DiscovToday. 2003 Dec. 15; 8(24):1128-37). It can be performed under a widerange of conditions (temperature, pH, solvents), the reaction is fastand the resulting triazoles are very stable and formed in high yields.

As used herein, a “polymer” is a molecule formed by monomers in whicheach monomer is covalently linked to other monomers.

The term “monomer” is used herein to refer to a molecule that has theability to combine with identical or other molecules in a process knownas polymerization. The polymerization reaction may be a dehydration orcondensation reaction (due to the formation of water (H₂O) as one of theproducts) where a hydrogen atom and a hydroxyl (—OH) group are lost toform H₂O and an oxygen molecule bonds between each monomer unit.

Examples of polymers suitable for use in this invention are polyethyleneoxide (PEO), polyethylene glycol (PEG), polyisopropylacrylamide(PNIPAM), polyacryl amide (PAM), polyvinyl alcohol (PVA),polyethylenimine (PEI), polyacrylic acid, polymethacrylate andpolyvinylpyrrolidone (PVP) polyvinyls, polyesters, polysiloxanes,polyamides, polyurethanes, polycarbonates, fluoropolymers, polyethylene,polystyrene, polybutadiene, polydimethylsiloxane (PDMS), polypropylene,polymethylmethacrylate, polytetrafluoroethylene and polyvinyl chloride(PVC).

Additional examples of suitable polymers include, but are not limitedto, those described in the references cited in this written descriptionand incorporated by reference herein. Nomenclature pertinent tomolecular structures, as well as description of monomers and side chainstructures useful for the present invention can be found in U.S. PatentPublication No. U.S. 2009/0011946, which is hereby incorporated byreference in its entirety.

As used herein, the term “polysaccharides” refers to polymericcarbohydrate structures, formed of repeating units (either mono- ordi-saccharides) joined together by glycosidic bonds. Polysaccharides ofthe invention are preferably linear, but may contain various degrees ofbranching. Additionally, polysaccharides are generally heterogeneous,containing slight modifications of the repeating unit. Examples ofpolysaccharides suitable for the invention include homopolysaccharidesor homoglycans, where all of the monosaccharides in a polysaccharide arethe same type, and heteropolysaccharies or heteroglycans, where morethan one type of monosaccharide is present. In exemplary embodiments,the polysaccharide is a starch, glycogen, cellulose, or chitin.

Polysaccharides of the invention have the general formula ofC_(X)(H₂O)_(y). In some embodiments, X is about 100 to about 100,000,about 200 to about 10,000, about 500 to about 5,000, or about 1,000 toabout 2,000. In another embodiment, polysaccharides have repeating unitsin the polymer backbone of about six-carbon monosaccharides and can berepresented by the general formula of (C₆H₁₀O₅), where n is about 30 toabout 100,000, about 200 to about 10,000, about 500 to about 5,000, orabout 1,000 to about 2,000.

As used herein, the terms “polynucleotide,” “oligonucleotide,” “nucleicacid” and “nucleic acid molecule” are used interchangeably herein torefer to a polymeric form of nucleotides of any length, and may compriseribonucleotides, deoxyribonucleotides, analogs thereof, or mixturesthereof. This term refers only to the primary structure of the molecule.Thus, the term includes triple-, double- and single-strandeddeoxyribonucleic acid (“DNA”), as well as triple-, double- andsingle-stranded ribonucleic acid (“RNA”) or RNA/DNA hybrids. It alsoincludes modified, for example by alkylation, and/or by capping, andunmodified forms of the polynucleotide. More particularly, the terms“polynucleotide,” “oligonucleotide,” “nucleic acid” and “nucleic acidmolecule” include polydeoxyribonucleotides (containing2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), includingtRNA, rRNA, siRNA, and mRNA, whether spliced or unspliced, any othertype of polynucleotide which is an N- or C-glycoside of a purine orpyrimi dine base, locked nucleic acids (LNA) which have one or more RNAnucleotides with a modified ribose that has an extra bridge connectingthe 2′ oxygen and 4′ carbon (Petersen M. et al. Trends Biotechnol. 21(2): 74-81 (2003)) and other polymers containing nonnucleotidicbackbones, for example, polyamide (e.g., peptide nucleic acids (PNAs))and polymorpholino (commercially available from the Anti-Virals, Inc.,Corvallis, Oreg., as Neugene) polymers, and other synthetic nucleic acidpolymers providing that the polymers contain nucleobases in aconfiguration which allows for base pairing and base stacking, such asis found in DNA and RNA. The term nucleotides include hybrids thereof,for example between PNAs and DNA or RNA, and also include known types ofmodifications, for example, labels, alkylation, “caps,” substitution ofone or more of the nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoramidates, carbamates,etc.), with negatively charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), and with positively charged linkages (e.g.,aminoalkylphosphoramidates, aminoalkylphosphotriesters), thosecontaining pendant moieties, such as, for example, proteins (includingenzymes (e.g. nucleases), toxins, antibodies, signal peptides,poly-L-lysine, etc.), those with intercalators (e.g., acridine andpsoralen), those containing chelates (e.g., metals, radioactive metals,boron, oxidative metals, etc.), those containing alkylators, those withmodified linkages (e.g., alpha anomeric nucleic acids, etc.).

Oligonucleotides of defined sequences are used for a variety of purposesin the practice of the invention. Methods of making oligonucleotides ofa predetermined sequence are well-known. See, e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual (2nd ed. 1989) and F. Eckstein(ed.) Oligonucleotides and Analogues, 1st Ed. (Oxford University Press,New York, 1991). Solid-phase synthesis methods are preferred for botholigoribonucleotides and oligodeoxyribonucleotides (the well-knownmethods of synthesizing DNA are also useful for synthesizing RNA).Oligoribonucleotides and oligodeoxyribonucleotides can also be preparedenzymatically.

As used herein, the term “polypeptides” refers to a polymer formed fromthe linking, in a defined order, of preferably, a-amino acids, D-,L-amino acids and combinations thereof. The terms “peptides,”“oligopeptides,” and “proteins” are included within the definition ofpolypeptide. The term includes polypeptides containingpost-translational modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations, and sulphations. Inaddition, protein fragments, analogs (including amino acids not encodedby the genetic code, e.g. homocysteine, ornithine, D-amino acids, andcreatine), natural or artificial mutants or variants or combinationsthereof, fusion proteins, derivatized residues (e.g. alkylation of aminegroups, acetylations or esterifications of carboxyl groups) and the likeare included within the meaning of polypeptide. The link between oneamino acid residue and the next is referred to as an amide bond or apeptide bond. The terms do not refer to a specific length of thepolypeptide.

In some embodiments, the elongated region of one or both probes comprisea non-biological hydrophilic polymer, such as polyethylene oxide (PEO),polyethylene glycol (PEG), polyisopropylacrylamide (PNIPAM),polyacrylamide (PAM), polyvinyl alcohol (PVA), polyethylenimine (PEI),polyacrylic acid, polymethacrylate and polyvinylpyrrolidone (PVP), or acombination thereof

In preferred embodiments, the elongated region of one or both probesand/or the target analyte comprises a biomolecule, such as apolysaccharide, polynucleotide, or a polypeptide, or a combinationthereof.

A preferred embodiment is a method of detecting a target analyte in asample. In this method, a complex is provided, the complex formed from:a first probe coupled to a detectable piece and bound to said analyte ifpresent, and a second probe coupled to a solid support and bound to saidanalyte if present, so that only if the target analyte is present in thesample, the detectable piece is directly or indirectly coupled to thesolid support via a complex formed by the target analyte and the firstand second probes, and wherein the complex optionally comprises anelongated region. It will be understood that the provided complex mayhave been formed in any possible manner and order of steps. It will alsobe clear that the person(s) who carries out any of the complex-formationsteps may, but need not be, the person who performs the subsequent stepsin the process, i.e., contacting the complex with a disruptor, applyingforce.

Preferably, the first probe, the second probe, and the target analytecomprise nucleic acids or oligonucleotides. More preferably, the firstprobe and second probe each comprise a region for binding the targetanalyte. The first probe couples to a detectable piece. The second probecouples to the solid support.

In preferred embodiments, if the target analyte is a nucleic acid, thenucleic acid target can form base pairs with unpaired nucleotides in thefirst probe and with unpaired nucleotides in the second probe. In anaspect of this embodiment, the first probe further comprises a regionfor coupling to the detectable piece. For example, the first probe cancomprise nucleotides that do not pair with the target analyte, aprotein, a peptide, an antigen covalently attached to the 5′ or 3′ endof a nucleic acid, or an amine group.

In another preferred aspect, the target analyte is not a nucleic acid.When the target analyte is not a nucleic acid, the first and secondprobes preferably comprise an antibody or an aptamer.

In preferred embodiments, the complexes are exposed to a disruptor. Thedisruptor is selected so that it preferentially uncouples detectablepieces that are coupled to the solid support via a target complex whiledetectable pieces which are coupled without the target complex are notaffected. After exposing the complexes to the disruptor, a force may beapplied to ensure that uncoupled beads leave their location. This forceis preferentially viscous drag and is applied using solution flow.Several other types of forces are suitable, such as gravity, magneticforce and electric force. The number of detectable pieces that leavetheir initial location or the detectable region is quantified. Thisquantification can be made in several ways: counting the total number ofdetectable pieces before and after the application of the disruptor,identifying each individual detectable piece and counting the ones thatremain attached/tethered after the application of the disruptor,collecting and counting the detectable pieces that leave the detectableregion, measuring the intensity of a signal generated by the detectablepieces before and after the application of the disruptor wherein thesignal correlates with the number of detectable pieces. In the lattercase, the conversion to number of detectable pieces may not be done andthe reported signal is just the difference between the intensity beforeand after the application of the disruptor. Examples of suitable signalsfor intensity measurements include: light intensity generated byfluorescent or color molecules, and electrical current induced in asolenoid by magnetic particles.

In one embodiment, the complex formed by the target analyte and thefirst and the second probe may comprise an elongated region. In apreferred embodiment, the elongated region of the complex formed by thetarget analyte and the first and the second probe comprisesdouble-stranded DNA having a total length of more than about 300 basepairs, preferably ranging from about 500 base pairs to about 300,000base pairs, for example, from about 3,000 base pairs to about 150,000base pairs.

In a preferred aspect, the target analyte is a nucleic acid and thefirst and second probes bind to locations on the target that are atleast 300 nucleotides from each other. According to this aspect thetarget can be either double or single stranded. When the target issingle stranded, the force required to extend it is significantly higherthan the force required to extend a double stranded nucleic acid(Current Opinion in Structural Biology 2000, 10:279; Nucleic AcidsResearch 2014 (42), 3:2064). The force required to extend the singlestranded nucleic acid can be modified by changing solution properties,such as ionic strength and temperature, and/or adding a molecule thatbinds to the single strand.

When the target analyte is a nucleic acid molecule, exposure of thetarget analyte to the first and/or second probe is preferably conductedunder high stringency conditions. High stringency conditions favor thehybridization of nucleic acid molecules which are perfectlycomplementary or substantially perfectly complementary to singlestranded nucleic acids in the probe and make more unlikely the bindingof targets which are not perfectly complementary or substantiallyperfectly complementary. After exposure of the target solution to thefirst and/or second probe, washing or exposing the probes to a mediumwith high stringency can remove non-perfectly complementary molecules aswell. High stringency conditions occur at high temperature, low saltconcentration and high pH. Also, the presence of certain chemicals, suchas formamide, can increase the stringency of the solution. In anembodiment, exposure of the target to probes and washing, whenperformed, are conducted preferably at temperatures between 20° C. and70° C., ionic strength between 0.01 M and 1 M, and pH between 7 and 8.

Some methods of this invention contain “exposing” steps where theprobe(s), detectable pieces, and/or solid support are exposed to thesample or one another. These exposing steps can occur in any order, oreven simultaneously. For example, in one embodiment, reactants areexposed in the following order: a) the first and second probe areexposed to the target analyte, b) the first probe which comprises afirst end for coupling to the detectable piece is exposed to thedetectable piece c) the second probe which comprises a first end forcoupling to the solid support, is exposed to the solid support. If thesesteps are conducted under conditions that allow reactants to bind orcouple and if the target analyte is present in the sample, thedetectable piece is indirectly coupled to the solid support via acomplex formed by the target, and the first and second probes. However,in another embodiment of the present invention, the steps are conductedin reverse order (c-b-a). If these steps are conducted under conditionsthat allow reactants to bind or couple and if the target analyte ispresent in the sample, the detectable piece is indirectly coupled to thesolid support as before: via the first probe, the target analyte and thesecond probe. In another exemplary embodiment all the steps can beconducted simultaneously. If this single step is conducted underconditions that allow reactants to bind or couple and if the targetanalyte is present in the sample, the detectable piece is indirectlycoupled to the solid support in the same manner as the previous twoexamples: via the first probe, the target analyte and the second probe.Any order and/or combination of steps that are conducted simultaneouslywill have the same result, if each step is conducted under conditionsthat allow reactants to bind or couple.

The methods of this invention can include one or more washing steps. Awashing refers to an exchange of the solution that the solid support isin contact with. The washing can be used to apply a fluid drag force onthe detectable pieces such that uncoupled detectable pieces are removed,and only the detectable pieces which are coupled (specifically andnon-specifically) to the solid support are left on the solid support. Awashing step can also be used to replace the buffer in contact with thesolid support for a buffer of different stringency. For example, a highsalt buffer (>0.1 Molar Sodium Chloride) may be exchanged for one of lowsalt (<0.1 Molar Sodium Chloride). High stringency buffers and/or fluiddrag force can be used to denature complexes formed with analytes thatare not the target but resemble the target analyte and therefore arecapable of binding to the first and second probe.

In preferred embodiments, the detectable piece is a solid bead with adiameter between about 0.1 micrometer and about 20 micrometer, orbetween about 0.3 micrometer and about 5 micrometer. Preferred beadmaterials include: silica-based glasses, such as quartz andborosilicate; zirconium; and organic polymers, such as polystyrene,melamine resin and polyacrylonitrile.

In preferred embodiments, the detectable piece is a superparamagneticbead with a diameter between about 0.3 micrometers and about 5micrometers. Superparamagnetic beads are commonly used inbiotechnological applications. They consist of a polymer matrix thatcontains small (less than about 10 nanometers) particles of aferromagnetic material in it. The small size of the ferromagneticparticles makes them superparamagnetic. As a result, the beads aremagnetic only under the influence of an external magnetic field.

In some embodiments, the detectable piece is a quantum dot. A quantumdot is typically less than about 10 nanometers and made of semiconductormaterials that display quantum mechanical properties. As a result ofthese properties, the electronic characteristics of quantum dots arerelated to the size and shape of the individual crystal. Quantum dotsare fluorescent and the emission frequency increases as the size of thequantum dot decreases. Therefore, the color of quantum dots can becontrolled by their size.

In some embodiments, the detectable piece is a nanorod. Preferably, thelength of the nanorods is at least about 0.5 micrometers. Methods ofmaking nanorods or nanowires are known in the art. See for example, Hahmand Mieber, Nano Lett, 4, 51-54 (2004) (silicon nanorods); Li et al.,Appl. Phys. Lett. 4, 4014-1016 (2003) (In2O3 nanorods); Liu et al.,Phys. Ev. B. 58, 14681-14684 (1998) (Bismuth nanorods); Sun et al.,Appl. Phys. Lett. 74, 2803 (1999) (Nickel nanorods); Ji et al., J.Electrochem. Soc. 150, C523-528 (2003) (Au/Ag multilayers andmultisegment nanorods); Celedon et al., Nano Lett., 9, 1720-1725 (2009)(Pt/Ni multisegment nanorods); O'Brien et al., Adv. Mater. 18, 2379-2383(2006) (polymer nanorods); Liu et al. Nanotechnology 20, 415703 (2009)(superparamagnetic and ferromagnetic Ni nanorods).

In some embodiments, the detectable piece comprises a fluorescentmolecule. These molecules are known to those skilled in the art. Forexample, a fluorescent nucleic acid can be created in a PCR reactionwhere one of the deoxynucleotides in the reaction mix has a florescentlabel. A commonly used labeled deoxyadenosine triphosphate for thisprocedure is Fluorescein-12-dATP. Protocols to label nucleic acidmolecules are readily available (Nucl. Acids Res. (1994) 22 (16):3418;Nat Biotechnol. (2008) 26(3):317; Nat Biotechnol. (2000) 18 (2):233).Another example of fluorescent molecule is a single fluorophore, such asCy3 and other cyanines, and fluorescein.

In some embodiments, the detectable piece comprises an enzyme capable ofcatalyzing the formation of a detectable molecule. Examples of this typeof enzyme include horseradish peroxidase and alkaline phosphatase. Theseenzymes act on numerous substrates, including chromogenic andchemiluminescent substrates. Horseradish peroxidase produces a coloured,fluorimetric, or luminescent derivative of the labeled molecule whenincubated with a proper substrate, allowing it to be detected andquantified. Horseradish peroxidase catalyses the oxidation of luminol to3-aminophthalate via several intermediates. The reaction is accompaniedby emission of low-intensity light at 428 nm. An example of thisembodiment is a detectable piece comprising a branched nucleic acid,which can contains several enzymes (Nucl. Acids Res. (1997) 25 (15):2979)

In some embodiments, the probes and/or target may be labeled before orafter the application of force with at least two detectable pieces, onedetectable piece at one end of the probe-target complex, the other atthe other end. In one embodiment, the elongated region of the complexmay be labeled substantially along its length with fluorescentmolecules. For example, the elongated region may be a double strandedDNA that is labeled with a nucleic acid fluorescent dye, such as YOYO-1.The approximate length of the elongated region can be determined fromthe position of said particles after the application of force. In theseembodiments the discrimination of non-specific interaction is based onthe length of the elongated region. If the full length of the elongatedregion is observed, it means that the target analyte is present.Instead, if a fraction of the length of the elongated region isobserved, it means that the attachment to the solid support is vianon-specific interactions.

In an aspect according to some of the embodiments, a force is applied tothe detectable pieces. The force field acts on the detectable piece andpulls it away from its initial position (e.g. a magnetic field acting ona magnetic particle or a flow exerting a drag on a particle). The deviceis exposed to a sample in conditions such that if the target analyte ispresent, then a complex is formed by the target analyte and the firstand second probes. Consequently, when a force is applied, the detectablepiece that is associated with the complex will move a distance that is afunction of the length of the complex.

In preferred embodiments, the presence of the target analyte in thesample is indicated by detectable pieces that: i) suffer a displacementwithin a pre-determined range and ii) leave their initial location afterthe complex is exposed to a disruptor. In these embodiments, the signalgenerated by non-specific interactions is reduced as shown in Example 1and Example 2. The pre-determined displacement is given by the length ofthe complex elongated region. The requirement that pieces suffer adisplacement within a range removes from consideration detectable piecesthat are non-specifically attached and suffer a displacement that isdifferent than expected based on the length of the elongated region, forexample pieces that are directly attached to the solid support will movea distance that is less than the pre-determined range. However, somedetectable pieces that are non-specifically attached suffer adisplacement that is within the pre-determined range. This can happenwhen beads attach non-specifically to the elongated molecule (asexemplified by beads 202 and 302 in FIGS. 2A, 2B, and 3). Therequirement that pieces leave their initial location removes fromconsideration most detectable pieces that are non-specifically attached.However, some detectable pieces that are non-specifically attached canleave their initial location. This can happen, for example, when thenon-specific attachment is reversible. By combining requirements i) andii), the signal generated by non-specifically attached pieces is greatlyreduced.

In other embodiments, instead of applying a force and measuring theamount of the detectable piece displacement, the amount of Brownianmotion in the absence of force is measured. The amount of Brownianmotion of a detectable piece in the absence of force increases with thelength of the tether that couples the detectable piece to the solidsupport and can be used to estimate the length of the tether. Therefore,Brownian motion can be used, for example, to discriminate detectablepieces that are non-specifically attached to the solid support fromdetectable pieces that are attached to the solid support via a complexthat comprises an elongated molecule.

In one embodiment, the detection device is exposed to the sample inconditions such that the number of beads tethered by probes isproportional to the concentration of the target analyte. In this manner,the detectable signal is proportional to the concentration of the targetanalyte, thereby permitting the concentration of the target analyte inthe sample to be determined.

Preferred embodiments use probes having elongated regions of multipledifferent lengths, with each probe of a certain length having a regioncapable of binding a specific target analyte, in such a manner that eachprobe length is associated with a different target analyte. In thisembodiment, the approximate concentration of multiple target analytes ina sample can be determined in a single assay by measuring differentdisplacements of particles after application of force, grouping thembased on the amount of displacement and counting the number of particlesassociated with each possible displacement.

In related embodiments, the multiplexing capability is further increasedby modifying the probes having elongated regions in such a manner thateach of them may have more than one length and the change of length istriggered by an external agent. Examples of external agents that cantrigger a change of length are temperature, ionic strength, pH, force,an auxiliary molecule, such as a nucleic acid, an enzyme a detergent,etc. In these embodiments, the identity of a target is determined aftermeasuring the displacement of the detectable piece before and aftertriggering the change of length. The change of length can be triggeredmultiple times. For example, in an assay with 100 different probeshaving an elongated region, each probe specific to a different targetanalyte can be created by a set of probes that have 10 different lengthsbefore triggering the length change and wherein each probe experiencesone out of 10 possible different length changes upon triggering thelength change. An example of a probe with an elongated region having alength that can be changed by an external agent is a probe in which tworemote positions in the probe interact in such a manner that an internalloop is formed. In this case, the external agent can trigger the releaseor the formation of an internal loop. The characteristics of theexternal agent required to trigger the change of length are controlledby the characteristics of the interaction. For example, if theinteraction is the hybridization between nucleic acid molecules, thespecific sequence can be used to modulate the characteristics of thetriggering agent. An example of an external agent that can trigger therelease of an internal loop formed by hybridization between nucleic acidmolecules is an auxiliary nucleic acid complementary to one of themolecules in the hybridization region that holds an internal loop. Whenthe probe is exposed to this auxiliary nucleic acid, the auxiliarynucleic acid can displace one of the strands in the hybridizationregion, thereby releasing the loop. Another example of an external agentis an auxiliary nucleic acid that has a first and a second region,wherein the first region is complementary to a first region in a nucleicacid probe and the second region is complementary to a second region inthe probe. When the probe is exposed to this nucleic acid, the nucleicacid binds to the two regions in the probe which produces an internalloop. In related embodiments, the auxiliary molecules are proteins thatcan be used similarly to the nucleic acid described above with thepurpose of releasing or forming internal loops.

The surfaces and probes of the present invention may have a plurality ofdifferent analyte binding molecules attached to them, and as a result,the tethering of detectable pieces to the solid support could betriggered by a plurality of target analytes.

In one embodiment, a solid support may have an array of regions, eachregion comprising a second probe specific to a unique target analyte.Thus, exposure of the solid support to the sample captures differenttargets at different locations in the array. Therefore, detection ofdetectable piece displacement in a specific array region indicates thatthe corresponding target analyte is present in the sample. According tothis embodiment, a method is provided for creating a unique profile orfingerprint of a sample having any number of different target analytes(e.g., any of two through one thousand, or even more). As such, profilesfrom different samples can be stored in a database and/or compared fordiagnostic purposes for the detection of diseases or disorders.

Another embodiment uses multiple distinguishable particles, wherein eachdifferent detectable piece comprises a different first probe specific toa different molecule. For example, fluorescent beads of different colorsare functionalized with different antibodies, one antibody kind for eachbead color. In this manner, the specific target analytes present in thesample are identified by detecting the color of the tethered beads thatare displaced under a force. Alternatively, beads of different sizes ordistinguishable strings of fluorescent molecules or particles can beused (Nat. Biotech. 26, 317-325, 2008).

In a preferred aspect of some of the embodiments, the applied force isfluid drag. This type of force is generated by the flow of the liquidsolution in which the detectable piece is immersed. More precisely, thisforce is applied when there is a difference between the speed of theliquid and the speed of the detectable piece. This force is oftenparallel to the solid support, but it can have a component perpendicularto the solid support if the solid support is porous. In preferredembodiments, the particles are in proximity to the surface of the solidsupport and the flow is substantially parallel to the surface of thesolid support. In these embodiments, the speed of the fluid increasesaway from the surface of the solid support and not only produces alinear force substantially parallel to the surface of the solid supportbut also a torque. The terms “fluid drag” and “fluid drag force” areused to indicate the combination of both the linear force and torque,when it exists, experienced by the particles. In preferred embodiments,the particles have a diameter or length less than about 20 micrometersand the flow is laminar, with a Reynolds number less than about 1.Typically, the particles and/or molecule are inside a capillary tube andflow can be generated using a pump, such as a syringe pump, connected tothe capillary by a hose. Another way of generating a suitable flow is toexert an external pressure on the hose connected to the capillary. Thepressure moves a small amount of fluid into the capillary, whichdisplaces the particles away from the side of the capillary where thepressure was applied. Releasing the pressure in the hose displaces thebeads in the opposite direction.

In an aspect of some of the embodiments, the applied force on thedetectable piece is fluid buoyancy. This type of force is equal to theweight of the fluid displaced by the detectable piece and in thedirection opposite to the gravitational force.

In an aspect of some of the embodiments, the applied force is a magneticforce. In these embodiments, the particles are magnetic, such assuperparamagnetic beads. The force is a consequence of the presence of amagnetic field which can be generated with permanent magnets, such asiron or rare earth magnets, or electromagnets. The magnetic force can beused to pull the particles away from the glass surface, in such a waythat particles tethered via an elongated region are displaced to a planehigher than the particles non-specifically attached to the surface. Thistype of displacement can be detected optically using an imaging systemable to image planes parallel to the solid support. In this imagingsystem, this type of displacement produces a change in diffractionpattern when the detectable piece moves to a different focal plane.Alternatively, the magnetic force can be used to pull the particles in adirection parallel to the surface of the solid support. This type ofdisplacement is easily detected as a change of position of the particlesin the image using an optical system.

In another aspect of some of the embodiments, the applied force isgravitational. In these embodiments the direction of the force is alwaystoward the center of the earth and therefore its direction with respectto the solid support is determined by the orientation in space of thesolid support.

In another aspect of some embodiments, the applied force is centrifugal.In these embodiments, the particles are subjected to a motion thatchanges direction. Preferably, the motion is a rotational motion.

In another aspect of some of the embodiments, the applied force iselectrical. An electrical force is generated when at least twoelectrodes having different voltage are introduced in the solutiongenerating a voltage gradient.

In another aspect of some of the embodiments, the force is applied tothe target-probe complex using flow, a receding meniscus, or a voltagegradient.

In embodiments that use a force substantially parallel to the solidsupport, such as embodiments that apply fluid drag substantiallyparallel to the solid support, the force can remove non-specificallybound particles while not significantly reducing the signal becausebeing part of a complex with an elongated region reduces the forceexperienced by the target analyte. When force substantially parallel tothe solid support is applied on particles bound to the solid support,the tension on the tether decreases with tether length (Langmuir 1996,12(9): 2271). Therefore, non-specific interactions, which are normallytethers about 10 nanometers long, experience tensions that aresignificantly higher than the tension that a target bound in anelongated complex experience. This property of long tethers allows inembodiments of the present invention the removal of non-specificallybound particles without significantly affecting specifically boundparticles. Using complexes comprising an elongated region larger thanthe non-specific tethers present in a particular assay improves theselective removal of nonspecifically bound particles in that assay. Ifthe tethered detectable piece is a sphere of radius a touching the solidsupport that experiences a force parallel to the solid support, as shownin FIG. 8A, then the tension (T) in the tether (length L) as a functionof the horizontal force (F) can be calculated by balancing forces andtorques. FIG. 8B shows the value of the ratio of tension and horizontalforce (T/F) as a function of the ratio of tether length and detectablepiece radius (L/a). The tension in the tether dramatically increases fortether lengths that are less than half the radius of the particle. Forexample, for a tether that is 0.01 times the radius of the particle, thetension is 7 times higher than the horizontal force, while for a tetherthat is 2 times the radius of the particle, the tension is only 6%higher than the horizontal force. In embodiments that apply fluid dragsubstantially parallel to the solid support, the torque applied on thedetectable piece by the drag further increases the difference in thetension experienced by short versus long tethers.

The application of force to the complex formed by the target analyte andthe first and second probes either directly or indirectly through forceapplied to the detectable piece can increase the target specificity ofthe detection system by removing complexes where the target is not theexact binding partner of the binding regions in the probes. When thetarget is a nucleic acid, this situation takes place, in most cases,when the target is not perfectly complementary to a nucleic acid regionin the probes. The application of force is a novel form of hybridizationstringency. This stringency can be modulated by the configuration of thestructure formed by probes when they bind to the target. In particularfor nucleic acids, a probe can hybridize to a target in two main typesof configurations. In a first configuration, the axis of the duplex isin the direction of the force and the application of force tends todisrupt all the base pairs simultaneously. In a second configuration,the axis of the duplex is perpendicular to the direction of the forceand the application of force tends to disrupt base pair in a progressiveorder, starting with the ones closer to the point of force application.The stringency can be modulated by the amount of force applied (CurrentOpinion in Chemical Biology 2008, 12: 640, PNAS 2006 (103), 16:6190).

In some embodiments, the present invention may be incorporated into anassay as described in: International Patent Publication WO 2013/059044;United States Patent Application Publication US 2014/0099635 A1; UnitedStates Patent Application Publication US 2016/0258003 A1; and UnitedStates Patent application Publication US 2015/0307926 A1; the contentsof these publications are incorporated by reference herein.

In some embodiments of the present invention, the presence anddisplacement of the particles is detected using an imaging system,wherein the imaging system generates an image of the particles and/orthe probes-target complex that is detected by a sensing device. Theimage can be a regular image or a transformed representation of theobject such as a shadow. The imaging system consists of four maincomponents: illumination, specimen, image forming part, and a detector,which are sequentially positioned on the spatial path. An example of animaging system is the optical microscope, and in this case the imageforming part is the lens/lenses. Optical microscopes are well known bythose of skill in the art. Optical microscopes can visualize unstainedsamples using image contrast of scattering, absorption or phasecontrast, or stained samples with fluorescence or other scheme of lightemission. The light source employed in a microscope can be coherencelight source (such as laser) or incoherent source (such as LED or whitelight source). The lenses of a microscope can be a single lens, a seriesof lenses, or a compound lens which is usually called an objective. Amacroscope is another example of imaging system. The main differencebetween a macroscope and microscope is the lens/objective they use. Themicroscope lens usually has magnification equal or larger than 1×,meaning the size of image is larger than the object. That results in asmall field of view. The macroscope lens can have magnification smallerthan 1×, which allows for visualization of a large area. A lens-freeimaging system is another example of an imaging system. This type ofsystem uses a digital optoelectronic sensor array, such as a chargedcoupled device (CCD) or a CMOS chip to directly sample the lighttransmitted through a specimen without the use of imaging lenses betweenthe object and the sensor plane (Greenbaum, Nat. Methods 2012, 9, 9,889-895; Gurkan, U. A., et al., Biotechnol. J. 2011, 6, 138-149). Thelensless ultra wide-field cell monitoring array platform (LUCAS) (Ozcan,A. and Demirci, U. Lab Chip, 2008, 8, 98-106) is an example of this typeof microscopes. The LUCAS platform is based on recording the “shadowimages” of microscopic objects onto a sensor array plane. Microscopicobjects are uniformly illuminated with an incoherent light source or alaser. The cell shadow pattern is digitally recorded using a CCD or CMOSsensor array. A coherent imaging system is another example of imagesystem. This type of system uses the object to modulate the illuminationlaser beam and makes the modulated beam interfere with a reference laserbeam or the same illumination beam, then the interferential informationis recorded to reconstruct the information of the object. Digitalholography (Javidi, Opt. Lett., 2000, 25, 9, 610-612) and in-lineholography (Xu, PNAS, 2001, 98, 20, 11301-11305) are examples of thistechnique.

In some embodiments, the displacement of the magnetic particles isdetected from the induced current in a solenoid.

The present invention also is directed to a kit for detecting a targetanalyte in a sample, the kit comprising a) a particle; and b) a firstprobe capable of binding to the analyte, and to either a solid supportor to the particle, the first probe optionally comprising an elongatedregion that is longer than about 0.1 micrometers, preferably betweenabout 0.3 and about 100 micrometers long; c) a disruptor molecule, d)packaging material; and optionally e) instructions for use. In aparticular embodiment, the kit may further comprise a second probecapable of binding to the analyte at a location different than thelocation that the first probe binds to the analyte, and which also iscapable of binding to either a solid support or to the particle. Thesecond probe may optionally contain an elongated region that is longerthan about 0.1 micrometers, more preferably between about 0.3 and about100 micrometers long. If the first probe is for coupling to a solidsupport, the second probe is for coupling to the particle, and if thefirst probe is for coupling to the particle, the second probe is forcoupling to the solid support. The packaging material would be known toone of ordinary skill, and in certain embodiments would includeconventional bottles, vials, boxes, etc. The optional instructions foruse would preferably include conventional printed materials includedwithin the packaging material.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable methods and materials are described below. In caseof conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

EXAMPLES Example 1

This example demonstrates detection of 4 CFU of Candida albicans. Thereagents used are listed here with reference to FIG. 3: a first probe(304) is a DNA oligonucleotide with a first region of 26 nucleotides(nt) having a sequence complementary to a region of rRNA in the smallribosomal subunit of C. albicans, a second region with a random 20-ntsequence that is a toehold for a strand-displacement molecule, and a25-nt poly A region (only adenine bases); 1-micron superparamagneticbeads (301, 302, 303) functionalized with a 30 nt poly T oligonucleotide(only thymine bases) (305), note that only one oligonucleotide is shownon the surface of the bead, but in reality the surface of the bead iscovered with many oligonucleotides; a second probe (307) generated asdescribed below and a glass capillary (50 mm×4 mm×0.2 mm) functionalizedwith a 36-nt single stranded DNA oligonucleotide with a random sequence(310). The second probe was generated in the following manner: A plasmid(5.4 kbps) was linearized using the restriction enzyme, which cut theplasmid twice generating a large fragment with different 4 nt overhangsat each end, and a small fragment which was separated and discarded. Thelinearized plasmid was ligated using T4 ligase to two double strandedDNA fragments generated by hybridizing synthetic oligonucleotides. Thefirst fragment had one end with an overhang compatible to one of theoverhangs of the plasmid and the other end had a 26-nt overhangcomplementary to a region of the C. albicans rRNA in the small ribosomalsubunit different from the region where the first probe binds. Thesecond fragment had one end with an overhang compatible to the otheroverhang of the plasmid, and the other end had a 36-nt overhangcomplementary to the capture probe (309). A solution containing 1 CFU ofC. albicans per microliter was lysed by heating to 90° C. for 8 minutes.This lysate was used to spike a 50 microliter reaction that containedthe first and the second probes with an equivalent of 4 CFU. The mixturewas incubated at 65° C. for 30 minutes. Then, beads were added, and themixture was incubated at 50° C. for 10 minutes. The mixture was thenflowed into a glass capillary and beads were let sediment for 15minutes. Buffer solution was flowed to wash unbound beads. The beadsthat remained attached to the bottom of the capillary were imaged withflow in one direction (first image) and then imaged again after the flowwas reversed (second image). The optical system used to image the beadswas composed of a LED ring light, a telecentric lens and a camera. TheLED ring light provided a dark field illumination. The telecentric lens,which is popularly employed in machine vision, exhibited the samemagnification for objects at different distances. The lens had a largedepth of field of around 500 micrometers. The magnification of thesystem was 1:1, which helped to achieve a large field of view of 6.14 mmby 4.6 mm. According to the optical resolution of the lens, the imagesize of each particle was about 4 micrometers, which was sufficient toinvestigate the displacement of the particles. The camera used in thesystem had a 1/2.3 inch complementary metal oxide semiconductor (CMOS)chip, which had 4384 by 3288 pixels with pixel size of 1.4 micrometer.

A custom code written in Matlab was used to analyze the images anddetermine the displacement of most of the beads present in the field ofview. Beads that were too close to each other (less than about 6micrometers) were not included in the analysis. Comparing the positionof each bead in the first and second images allowed measuring thedisplacement of over 5,000 beads in each experiment with sub-micrometerresolution. The displacement of all the beads in the field of view wasused to generate a histogram of bead displacement. FIG. 4A shows thehistograms obtained in two experiments, one with 4 CFU of cells in thereaction (top) and one without cells (bottom). The histograms show 3peaks. Note that the peaks in the top histogram are displaced withrespect to the peaks in the bottom histogram, however the distancebetween peaks is the same. The peaks close to zero displacement (405)and (406) correspond to beads that do not move and are non-specificallyattached to the solid support, as shown in FIG. 3 (301). The peaks atabout 3 microns (401) and (403) are generated by beads that are coupledto the solid support via a positive control molecule. The peak at about4.5 microns (beads that suffer a displacement between 4.3 and 5.2microns) is generated by beads that are coupled to the solid support viaa complex formed by first probe, the target and the second probe. Thebeads that are non-specifically attached to the solid support with asmall tether (309) and peak (405) can be easily discriminated from thebeads in peak (402). Peak 402 has 3636 beads. However, the no-targetexperiment shows that not all the beads in (402) are coupled to thesolid support via a target complex. In fact, the region (404) contains807 beads (FIG. 4B). These beads are attached non-specifically at theend region of the second probe (302). The signal-to-background ratio is3636/807 4.5. We used the present invention to discriminate beadscoupled via a target complex from beads that despite presenting asimilar displacement are non-specifically attached to the second probe.A strand-displacement DNA oligonucleotide (377) was flowed into thecapillary at 5 micro-Molar concentration. After 4 minutes of gentle flowanother image was taken. The vicinity of the location of each of the3636 beads that formed peak 402 was analyzed in the new image to detectwhich beads remained at the location and which beads had left thelocation. We found that 2860 beads had left in the experiment at 4 CFU.We found also that only 3 beads left in the experiment with no target.The signal-to-background ratio is 2860/3≈953. Therefore, thesignal-to-background ratio was significantly improved by using thestrand-displacement DNA oligonucleotide.

Example 2

In another example of the present invention, an experiment with the samereagents as in Example 1 was conducted. The protocol was also the sameas in Example 1. The only difference was that instead of using astrand-displacement molecule as the disruptor, a degradation moleculewas used (10), specifically, an RNase enzyme was used. FIG. 5A showshistograms obtained in these experiments and the table of FIG. 5Bsummarizes the total number of beads in peaks (502) and peak (504), aswell as the number of beads from these peaks that left their locationafter exposure to RNase. Before disruption, the experiment with 4 CFUhad 5549 beads that displaced the expected distance (502), while in the“no-target” experiment had 427 beads that displaced the expecteddistance (503). Therefore, the signal-to-background ratio was5549/427≈13. The enzyme was flowed after obtaining the second image.Buffer with the enzyme was flowed at 0.5 gram per litter into thecapillary and incubated for 10 minutes, after 4 minutes of gently flowanother image was taken. As in Example 1, the vicinity of the locationof each bead in peak (502) of FIG. 5A was analyzed in this new image andthe number of beads that left their location was determined. We foundthat 4658 beads had left in the experiment at 4 CFU. We found also thatonly 15 beads left in the experiment with no target. Thesignal-to-background ratio is 4658/15≈310. Therefore, thesignal-to-background ratio was significantly improved by using the RNasedisruptor molecule.

Example 3

A 60 nucleotide (nt) synthetic RNA oligonucleotide target can bedetected using a first probe consisting of 30 nt single strandedoligonucleotide having a sequence complementary to the 3′ end of thetarget and a 30 nt poly A region (only adenine bases), 1-micronsuperparamagnetic beads functionalized with a 30 nt poly Toligonucleotide (only thymine bases), a second probe consisting of 30 ntsingle stranded oligonucleotide having a sequence complementary to the5′ end of the target and a 30 nt region with a sequence S1 and a glasscapillary functionalized with a DNA oligonucleotide with a sequencecomplementary to S 1. The first and second probes are mixed with asolution containing the target at 65° C. for 10 minutes. Then, the beadsare incubated at 50° C. for 10 minutes. The mixture is then flowed intoa glass capillary (50 mm×4 mm×0.2 mm) and let sediment. A buffersolution is flowed to wash unbound beads. A first image is taken and thebeads that remained attached to the bottom of the capillary are countedor estimated using an image analysis algorithm. A disruptor RNasemolecule in buffer at 0.5 gram per litter is flowed into the capillaryand incubated for 10 minutes. Buffer is flowed to removed uncoupledbeads. A second image is taken and the beads that remained attached tothe bottom of the capillary are counted or estimated using an imageanalysis algorithm. The beads that leave the capillary after theincubation with the disruptor molecule are the relevant signal. Toobtain this signal, the number of beads in the second image issubtracted from the number of beads in the first image.

Example 4

ELISA (enzyme-linked immunosorbent assay) is a plate-based assaytechnique designed for detecting and quantifying substances such aspeptides, proteins, antibodies and hormones. Other names, such as enzymeimmunoassay (EIA), are also used to describe the same technology. In anELISA, an antigen must be immobilized on a solid surface and thencomplexed with an antibody that is linked to an enzyme. Detection isaccomplished by assessing the conjugated enzyme activity via incubationwith a substrate to produce a measurable product. ELISAs are typicallyperformed in 96-well (or 384-well) polystyrene plates, which passivelybind antibodies and proteins. It is this binding and immobilization ofreagents that makes ELISAs so easy to design and perform. Having thereactants of the ELISA immobilized to the microplate surface makes iteasy to separate bound from non-bound material during the assay. Thisability to wash away nonspecifically bound materials makes the ELISA apowerful tool for measuring specific analytes within a crudepreparation. A detection enzyme or other tag can be linked directly tothe primary antibody or introduced through a secondary antibody thatrecognizes the primary antibody. It can also be linked to a protein suchas streptavidin if the primary antibody is biotin labeled. The mostcommonly used enzyme labels are horseradish peroxidase (HRP) andalkaline phosphatase (AP). Other enzymes have been used as well, butthey have not gained widespread acceptance because of limited substrateoptions. These include 0-galactosidase, acetylcholinesterase andcatalase. A large selection of substrates is available for performingELISA with an HRP or AP conjugate. The choice of substrate depends uponthe required assay sensitivity and the instrumentation available forsignal-detection (spectrophotometer, fluorometer or luminometer). ELISAscan be performed with a number of modifications to the basic procedure.The key step, immobilization of the antigen of interest, can beaccomplished by direct adsorption to the assay plate or indirectly via acapture antibody that has been attached to the plate. The antigen isthen detected either directly (labeled primary antibody) or indirectly(labeled secondary antibody). The most powerful ELISA assay format isthe sandwich assay. This type of capture assay is called a “sandwich”assay because the analyte to be measured is bound between two primaryantibodies—the capture antibody and the detection antibody. The sandwichformat is used because it is sensitive and robust.

No matter the type of ELISA assay performed, non-specific interactionsresult in the coupling of enzymes to the solid support that is notmediated by target molecule which generates background noise. Thisbackground noise reduces the sensitivity of the assay. According to thepresent invention, the background can be substantially eliminated byexposing the solid support to a disruptor which preferentially uncouplesenzymes molecules coupled via a target complex and then measuring thesignal intensity that is generated by the enzymes that leave theirinitial location. Alternatively, after exposing the solid support to adisruptor, the solid support can be washed, and the signal generated byenzymes that remain on the surface is measured to determine thebackground noise which can be subtracted to the initial signal intensityto determine the real signal.

Example 5

A standard branched DNA assay begins with a solid support functionalizedwith single stranded DNA molecules known as capture DNA. Next, anextender DNA molecule is added. Each extender has two domains; one thathybridizes to the capture DNA molecule and one that is capable ofbinding to the target. Once the capture and extender molecules are inplace and they have hybridized, the sample can be added. Targetmolecules in the sample will bind to the extender molecule. This resultsin a base peppered with capture oligonucleotides, which are hybridizedto extender probes, which in turn are hybridized to target molecules. Atthis point, signal amplification takes place. A label extender DNAmolecule is added that has two domains (similar to the first extender).The label extender hybridizes to the target and to a pre-amplifiedmolecule. The preamplifier molecule has two domains. First, it binds tothe label extender and second, it binds to the amplifier molecule. Anexample amplifier molecule is an oligonucleotide bound to the enzymealkaline phosphatase.

Non-specific interactions result in the coupling of amplifier moleculesto the solid support that is not mediated by target molecule whichgenerates background noise. This background noise reduces thesensitivity of the assay. According to the present invention, thebackground can be substantially eliminated by exposing the solid supportto a disruptor which preferentially uncouples amplifier moleculescoupled via a target complex and then measuring the signal intensity ofthe amplifiers that leave their initial location. Alternatively, afterexposing the solid support to a disruptor, the solid support can bewashed, and the signal generated by amplifiers that remain on thesurface is measured to determine the background noise which can besubtracted to the initial signal intensity to determine the real signal.

Example 6

Lateral flow tests also known as lateral flow immunochromatographicassays, are simple paper-based devices intended to detect the presence(or absence) of a target analyte in liquid sample (matrix) without theneed for specialized and costly equipment, though many lab basedapplications exist that are supported by reading equipment. Typically,these tests are used for medical diagnostics either for home testing,point of care testing, or laboratory use. A widely spread and well-knownapplication is the home pregnancy test. The technology is based on aseries of capillary beds, such as pieces of porous paper,micro-structured polymer, or sintered polymer. Each of these elementshas the capacity to transport fluid (e.g., urine) spontaneously. Thefirst element (the sample pad) acts as a sponge and holds an excess ofsample fluid. Once soaked, the fluid migrates to the second element(conjugate pad) in which the manufacturer has stored the so-calledconjugate, a dried format of bio-active particles (see below) in asalt-sugar matrix that contains everything to guarantee an optimizedchemical reaction between the target molecule (e.g., an antigen) and itschemical partner (e.g., antibody) that has been immobilized on theparticle's surface. While the sample fluid dissolves the salt-sugarmatrix, it also dissolves the particles and in one combined transportaction the sample and conjugate mix while flowing through the porousstructure. In this way, the analyte binds to the particles whilemigrating further through the third capillary bed. This material has oneor more areas (often called stripes) where a third molecule has beenimmobilized by the manufacturer. By the time the sample-conjugate mixreaches these strips, analyte has been bound on the particle and thethird ‘capture’ molecule binds the complex. After a while, when more andmore fluid has passed the stripes, particles accumulate and thestripe-area changes color. Typically, there are at least two stripes:one (the control) that captures any particle and thereby shows thatreaction conditions and technology worked fine, the second contains aspecific capture molecule and only captures those particles onto whichan analyte molecule has been immobilized. After passing these reactionzones the fluid enters the final porous material, the wick, that simplyacts as a waste container. In principle, any colored particle can beused, however latex (blue color) or nanometer sized particles of gold(red color) are most commonly used. The gold particles are red in colordue to localized surface plasmon resonance. Fluorescent or magneticlabeled particles can also be used, these require the use of anelectronic reader to assess the test result. Lateral Flow Tests canoperate as either competitive or sandwich assays. In sandwich assays, asthe sample migrates along the assay, it first encounters a conjugate,usually colloidal gold, which is labelled with antibodies specific tothe target analyte. If the target analyte is detected within the samplethe conjugate antibodies will bind and subsequently reach the test linewhich also contains antibodies specific to the target. Once the samplereaches the test line and the target analyte is present a visual change,normally a line appearing, will occur allowing the test to be read as apositive. Most sandwich assays also have a control line which willappear regardless of whether or not the target analyte is present. Therapid, low-cost sandwich-based assay is commonly used for home pregnancytests which detects for human chorionic gonadotropin, hCG, in the urineof women.

In competitive assays, the sample first encounters colored particleswhich are labelled with the target analyte or an analogue. The test linecontains antibodies to the target/its analogue. Unlabeled analyte in thesample will block the binding sites on the antibodies preventing uptakeof the colored particles. The test line will show as a colored band innegative samples.

In both sandwich and competitive lateral flow assays, non-specificinteractions result in the coupling of particles to the solid supportthat is not mediated by target molecule which generates backgroundnoise. This background noise reduces the sensitivity of the assay.According to the present invention, the background can be substantiallyeliminated by exposing the solid support to a disruptor whichpreferentially uncouples particles coupled via a target complex and thendetecting/measuring the particles that leave their initial location.Alternatively, after exposing the solid support to a disruptor, thesolid support can be washed, and the particles that remain on thesurface are detected/measured to determine the background noise whichcan be subtracted to the initial signal intensity to determine the realsignal.

Example 7

A DNA microarray (also commonly known as DNA chip or biochip) is acollection of microscopic DNA spots attached to a solid surface. DNAmicroarrays can be used to measure the expression levels of largenumbers of genes simultaneously or to genotype multiple regions of agenome. Each DNA spot can contain as little as 1 picomole (10⁻¹² moles)of a specific immobilized DNA oligonucleotide (capture oligonucleotide).These can be a short section of a gene or other DNA element that areused to hybridize a cDNA or cRNA (also called anti-sense RNA) in asample (target) under high-stringency conditions. Probe-targethybridization is usually detected and quantified by detection offluorophore-, silver-, or chemiluminescence-labeled targets to determinerelative abundance of nucleic acid sequences in the target.

Labeled nucleic acids in the sample that are not complementary to thecapture oligonucleotides in a spot couple to the solid support at thatspot via non-specific interactions which generates background noise.This background noise reduces the sensitivity of the assay. According tothe present invention, the background can be substantially eliminated byexposing the solid support to a disruptor which preferentially uncoupleslabeled nucleic acids coupled via a target complex and thendetecting/measuring the labeled nucleic acids that leave their initiallocation. Alternatively, after exposing the solid support to adisruptor, the solid support can be washed, and the labeled nucleicacids that remain on the surface are detected/measured to determine thebackground noise which can be subtracted to the initial signal intensityto determine the real signal.

What is claimed is:
 1. A method of detecting a target analyte in asample, the method comprising: a) providing at least one detectablepiece coupled to a solid support via a complex formed by the targetanalyte and a first and a second probe, wherein: i) the first probe iscoupled to the detectable piece and bound to said analyte if present,and ii) the second probe is coupled to the solid support and bound tosaid analyte if present, so that only if the target analyte is presentin the sample, the detectable piece is directly or indirectly coupled tothe solid support at an initial location via the complex, wherein thecomplex comprises an elongated region that is at least 100 nanometers inlength; b) either applying a force to the detectable piece and measuringthe displacement of the detectable piece or measuring the amount ofBrownian motion of the detectable piece; c) exposing the complex to adisruptor that is capable of uncoupling the detectable piece from thesolid support; d) optionally applying a force to the detectable piece;and e) detecting if the detectable piece was uncoupled from the solidsupport by the disruptor; wherein the presence of the target in thesample is indicated by detectable pieces that: i) suffer a displacementor Brownian motion within a pre-determined range and ii) are uncoupledfrom the solid support by the disruptor.
 2. The method of claim 1,wherein step e) is conducted by determining if the detectable piece leftthe initial location on the substrate where it was coupled by thecomplex.
 3. The method of claim 1, further comprising the step ofestimating the concentration of the target analyte based on the numberof detectable pieces that are uncoupled from the solid support by thedisruptor.
 4. The method of claim 1, wherein the first probe comprises afirst nucleic acid that binds to a first region of the target analyteand the second probe comprises a second nucleic acid that binds to asecond region of the target analyte.
 5. The method of claim 1, whereinthe first probe comprises a first antibody or a first aptamer that bindsto a first region of the target analyte and the second probe comprises asecond antibody or second aptamer that binds to a second region of thetarget analyte.
 6. The method of claim 1, wherein the target comprises aprotein, the disruptor comprises an enzyme capable of degrading thetarget analyte, and the first and second probes do not compriseproteins.
 7. The method of claim 5, wherein the disruptor comprisesproteinase K.
 8. The method of claim 1, wherein the target analytecomprises RNA, the disruptor comprises an enzyme capable of degradingthe target analyte, and the first and second probes are not degradableby the disruptor.
 9. The method of claim 7, wherein the disruptorcomprises RNase H, RNase A, RNase III, RNase L, RNase P, RNase PhyM,RNase T1, RNase T2, RNase U2, or RNase V.
 10. The method of claim 1,wherein the target analyte comprises a nucleic acid and the disruptorcomprises an endonuclease or exonuclease enzyme.
 11. The method of claim9, wherein the enzyme comprises a restriction enzyme.
 12. The method ofclaim 1, wherein the disruptor comprises a nucleic acid that canhybridize to the target analyte in a region where the target analytehybridizes to the first or second probe.
 13. The method of claim 1,wherein the disruptor comprises a nucleic acid that can hybridize to thefirst probe or to the second probe.
 14. The method of claim 1, whereintwo or more different target analytes are detected by using a differentcombination of first and second probe for each target analyte, whereineach target forms a different target complex with the probes that bindto it, and wherein a disruptor disrupts the target complex of one ormore target analytes.
 15. The method of claim 13, wherein the disruptorcomprises two or more different disruptors that are sequentiallycontacted with the complexes and the detectable pieces are uncoupledfrom the solid support by the disruptor are quantified after eachdisruptor is contacted with the complexes.
 16. The method of claim 1,which further comprises the step of coupling the first probe to thedetectable piece.
 17. The method of claim 1, which further comprises thestep of coupling the second probe to the solid support.
 18. The methodof claim 1, which further comprises the steps of exposing the sample tothe first probe and exposing the sample to the second probe.
 19. Themethod of claim 17, wherein the further steps are performed in eitherorder, or simultaneously.
 20. The method of claim 18, which furthercomprises a step of exposing the sample to a detectable piece.
 21. Themethod of claim 19, further comprising a washing step after one or moreof the steps.
 22. The method of claim 20, further comprising a lysingstep.
 23. The method of claim 1, wherein the force applied to thedetectable piece comprises a magnetic force, a fluid drag force, anelectrical force or a centrifugal force.
 24. The method of claim 1,wherein the detectable piece comprises a particle.
 25. The method ofclaim 23, wherein the particle is magnetic.
 26. The method of claim 24,wherein the magnetic detectable piece is superparamagnetic.
 27. Themethod of claim 1, wherein the detectable piece is fluorescent.
 28. Themethod of claim 1, wherein the detectable piece is detected using animaging system with a lens, with a lens-free microscope, or with acoherent imaging technique.
 29. The method of claim 3, which furthercomprises controlling the temperature of the sample to producedenaturation of double stranded nucleic acids in the sample and/orspecific hybridization of nucleic acids in the sample to the first andsecond probes.
 30. The method of claim 3, wherein the sample isinitially treated with an exonuclease enzyme to convert double strandednucleic acids into single stranded nucleic acids.
 31. The method ofclaim 1, wherein the target analyte comprises a protein, carbohydrate,lipid, hormone, steroid, toxin, vitamin, hapten, metabolite, drug, or acombination thereof.
 32. A method of detecting a target analyte in asample, the method comprising: a) providing at least one detectablepiece coupled to a solid support via a complex formed by the targetanalyte and a first and a second probe, wherein: i) the first probe iscoupled to the detectable piece and bound to said analyte if present,and ii) the second probe is coupled to the solid support and bound tosaid analyte if present, so that only if the target analyte is presentin the sample, the detectable piece is directly or indirectly coupled tothe solid support via the complex, b) optionally detecting the presenceof the detectable piece; c) exposing the complex to a disruptor that iscapable of uncoupling the detectable piece from the solid support,wherein the disruptor comprises a strand-displacement molecule capableof dissociating one or more nucleic acid duplexes formed between thetarget and a probe, or the disruptor comprises a degradation moleculecapable of breaking one or more covalent bonds of the target analyte; d)optionally applying a force to the detectable piece; and e) detecting ifany of the detectable pieces was uncoupled from the solid support by thedisruptor; wherein a detectable piece that is indirectly bound to theanalyte is likely to be uncoupled from the solid support by thedisruptor, whereas a detectable piece that is not indirectly bound tothe analyte is unlikely to be uncoupled from the solid support by thedisruptor.